Detection of SARS-CoV-2 spike antibody using electrochemical biosensing based on hydrogen bonding

The coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), originated and spread from Wuhan, which is the capital of China’s Hubei Province. As of November 22, 2021, over 258 million cases of COVID-19 have been reported, which have resulted in more than 5.15 million deaths globally. COVID-19, therefore, continues to pose severe threats for public health systems and economies around the world.  

Study: Electrochemical biosensing platform based on hydrogen bonding for detection of the SARS-CoV-2 spike antibody. Image Credit: anyaivanova / Shutterstock.com

Advantages and disadvantages of RT-PCR

The real-time polymerase chain reaction (RT-PCR) assay has remained the most prominent technique to diagnose COVID-19. However, other potentially useful detection assays are currently being studied, some of which include enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), lateral flow immunoassay (LFIA), ultraviolet (UV)–visible spectroscopy, clustered regularly interspaced short palindromic repeats (CRISPR), loop-mediated isothermal amplification (LAMP), hematological parameters, computed tomography (CT) imaging, plasmonic sensors, and electrochemical biosensors. These methods have the potential to become improved testing methods given their simplicity, rapidity, sensitivity, and accuracy.

RT-PCR is the most commonly used diagnostic test for COVID-19 due to its standardization, good sensitivity, and selectivity. However, RT-PCR is an expensive, labor-intensive, and time-consuming technique that and requires specially trained personnel for execution and, as a result, remains exclusive to laboratory-based medical institutions. Another major disadvantage associated with RT-PCR is a high false-negative ratio that ranges between 20–67%, depending on the time since infection.

The utility of electrochemical biosensing techniques

By contrast, electrochemical biosensing methods are simple, rapid, cost-effective, robust, and highly sensitive, and selective for diagnosing COVID-19. Furthermore, these methods can also detect the whole virus, the antibody produced in the body, as well as their specific fragments and proteins.

Antigen-based electrochemical methods based on viral ribonucleic acid (RNA) detection require seven hours and 29 hours for sensor preparation and 40 minutes and three hours for measurement to provide a Limit of Detection (LOD) of 6,900 copies/mL and 200 copies/mL, respectively. Both antigen- and antibody-based electrochemical methods use either a spike protein or nucleocapsid protein to diagnose COVID-19.

Previous studies have shown the possibility of getting a LOD of 0.01 ag/mL for the spike antibody in synthetic media using gold clusters, cysteamine, glutaraldehyde, and the SARS-CoV-2 spike antigen-modified glassy carbon electrode. As a continuation to this, Turkish researchers recently published a study in the journal Analytical and Bioanalytical Chemistry demonstrating a rapid electrochemical biosensing platform based on gold clusters (Au), and mercaptoethanol (CysOH) bovine serum albumin (BSA)-modified glassy carbon electrodes (GCE) to detect the SARS-CoV-2 spike antibody.

About the study

Initially, the Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein, influenza A spike protein, and Streptococcus pneumoniae antigen were used to examine the produced biosensor’s selectivity by separately immobilizing them on CysOH/Au/GCE electrodes and blocking them with BSA. The produced platforms were denoted as BSA/M-S-gene/CysOH/Au/GCE, BSA/InfA-S-gene/CysOH/Au/GCE, and BSA/Pneu/CysOH/Au/GCE, respectively.

As a consequence, the fabricated platforms showed no significant response to 1 fg/mL of the SARS-CoV-2 spike antibody. The interference effects of various anions, enzymes, and compounds that could be present in saliva were investigated in the presence of as little as a 1 fg/mL concentration of the SARS-CoV-2 spike antibody with a criterion to mark a 5% variation in the peak height for evaluation. The results were indicative of good selectivity of the method.

Saliva and oropharyngeal swab samples were collected from six healthy individuals. Ten fg of the SARS-CoV-2 spike antibody was added to half of the samples for each 5 μL, while the other half were left non-spiked. All samples were analyzed to determine the SARS-CoV-2 spike antibody via external calibration by depositing 5 μL of the sample on the surface of BSA/S-gene/CysOH/Au/GCE electrodes without any pre-processing.

During the detection of the SARS-CoV-2 spike antibody in spiked-real samples, the anodic signal of the produced biosensor at 0.85 V decreased as the amount of the SARS-CoV-2 spike antibody increased.

In 35 minutes, the biosensing platform could detect 0.03 fg/mL of the SARS-CoV-2 spike antibody in synthetic media and spiked-saliva or -oropharyngeal swab samples. The method thus showed a linear response to the increase in the concentration of SARS-CoV-2 spike antibody from 0.1 fg/mL to 10 pg/mL.

The cross-reactivity studies with spike antigens of MERS-CoV, influenza A, and the antigen of pneumonia confirmed the excellent selectivity of the proposed method. The developed method was compared with the LFIA method in terms of sensitivity and was found to be approximately 109 times more sensitive.

Relative standard deviation values were calculated to be 7.55%, 3.79%, and 5.23% for 1 fg/mL, 100 fg/mL, and 10 pg/mL of the SARS-CoV-2 spike antibody, respectively, thus indicating good reproducibility. The stability and robustness of the assay were assessed by storing the biosensor in an argon atmosphere by measuring the peak height at the end of six consecutive five-day periods at 4 °C, 25 °C, and 37 °C, respectively.

These stability tests showed no significant difference when stored at 4 °C versus 25 °C. By contrast, on day 30, the signal had preserved at least 84.5% of the signal from day 1, even when stored at 37 °C. These results indicate the exceptional stability and robustness of BSA/S-gene/CysOH/Au/GCE.

Implications

The rapid, inexpensive, sensitive, selective,m and accurate biosensing platform developed for the voltammetric determination of the SARS-CoV-2 spike antibody in spiked-saliva and -oropharyngeal swab samples did not require any sample pre-processing.

Moreover, the biosensor was also developed easily, with the shortest preparation time among prominent electrochemical biosensing methods based on antigen- or antibody-protein reported in scientific literature thus far. Furthermore, the current device had an analysis time of  approximately35 mins, which is significantly shorter than existing RT-PCR methods. Moving forward, BSA/S-gene/CysOH/Au/GCE could be easily fabricated and provided as a ready-to-use kit on a commercial scale by using disposable screen-printed electrodes.

Journal reference:
Sreetama Dutt

Written by

Sreetama Dutt

Sreetama Dutt has completed her B.Tech. in Biotechnology from SRM University in Chennai, India and holds an M.Sc. in Medical Microbiology from the University of Manchester, UK. Initially decided upon building her career in laboratory-based research, medical writing and communications happened to catch her when she least expected it. Of course, nothing is a coincidence.

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